Targeted sequencing of biomolecules by pulling through a liquid-liquid interface with an atomic force microscope

ABSTRACT

A mechanism is provided for sequencing a biopolymer. The biopolymer is traversed from a first medium to a second medium. The biopolymer includes bases. As the biopolymer traverses from the first medium to the second medium, different forces are measured corresponding to each of the bases. The bases are distinguished from one another according to the different measured forces which are measured for each of the bases.

DOMESTIC PRIORITY

This application is a continuation of U.S. Non-Provisional applicationSer. No. 13/915,430, entitled “TARGETED SEQUENCING OF BIOMOLECULES BYPULLING THROUGH A LIQUID-LIQUID INTERFACE WITH AN ATOMIC FORCEMICROSCOPE”, filed Oct. 27, 2014 which is a continuation of U.S.Non-Provisional application Ser. No. 13/899,728, entitled “TARGETEDSEQUENCING OF BIOMOLECULES BY PULLING THROUGH A LIQUID-LIQUID INTERFACEWITH AN ATOMIC FORCE MICROSCOPE”, filed May 22, 2013, both of which areincorporated herein by reference in their entirety.

BACKGROUND

The present invention relates to sequencing, and more specifically tosequencing by pulling molecules (at a constant rate) through one mediumto another medium.

Recently, there has been growing interest in applying nanopores assensors for rapid analysis of biomolecules such as Deoxyribonucleic acid(DNA), Ribonucleic acid (RNA), proteins, etc. Special emphasis has beengiven to applications of nanopores for DNA sequencing, as the technologywith the potential to reduce the cost of sequencing below $1000 perhuman genome.

Nanopore sequencing is a method for determining the order in whichnucleotides occur on a strand of DNA. A nanopore is simply a small holeof the order of several nanometers in internal diameter. The theorybehind nanopore sequencing has to do with what occurs when the nanoporeis immersed in a conducting fluid and an electric potential (voltage) isapplied across it: under these conditions a slight electric current dueto conduction of ions through the nanopore can be measured, and theamount of current is very sensitive to the size and shape of thenanopore. If single bases or strands of DNA pass (or part of the DNAmolecule passes) through the nanopore, this can create a change in themagnitude of the current through the nanopore. Other electrical oroptical sensors can also be put around the nanopore so that DNA basescan be differentiated while the DNA passes through the nanopore.

SUMMARY

According to an embodiment, a method of sequencing a block copolymer isprovided. The method includes traversing the block copolymer from afirst medium to a second medium. The block copolymer comprises firstblocks and second blocks. The method includes measuring a first measuredforce when the first blocks traverse from the first medium to the secondmedium, and measuring a second measured force when the second blockstraverse from the first medium to the second medium, where the firstmeasured force is different from the second measured force. The methodincludes identifying the first blocks of the block copolymer based onmeasuring the first level of force and identifying the second blocks ofthe block copolymer based on measuring the second level of force.

According to an embodiment, a method of sequencing a biopolymer isprovided. The method includes traversing the biopolymer from a firstmedium to a second medium, where the biopolymer includes bases. Themethod includes measuring different force levels corresponding to eachof the bases as the biopolymer traverses from the first medium to thesecond medium and distinguishing the bases from one another according tothe different measured forces measured for each of the bases.

Additional features and advantages are realized through the techniquesof the present invention. Other embodiments and aspects of the inventionare described in detail herein and are considered a part of the claimedinvention. For a better understanding of the invention with theadvantages and the features, refer to the description and to thedrawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The subject matter which is regarded as the invention is particularlypointed out and distinctly claimed in the claims at the conclusion ofthe specification. The forgoing and other features, and advantages ofthe invention are apparent from the following detailed description takenin conjunction with the accompanying drawings in which:

FIG. 1A illustrates an example setup for measuring the hydration andsolvophobic force of a polymer strand according to a first embodiment.

FIG. 1B illustrates a graph of the measured force versus molecularextension (F-x) curve according to the first embodiment.

FIG. 2 illustrates a method for measuring the solvophobic force andsolvophobic energy of the polymer molecule in the bad solvent to goodsolvent boundary according to the first embodiment.

FIG. 3A illustrates the example setup for measuring the solvophobicforce and energy of a diblock polymer strand and its nanoscalecomposition according to a second embodiment.

FIG. 3B illustrates a graph of the measured force versus molecularextension (F-x) curve according to the second embodiment.

FIG. 3C illustrates the second plateau region in more detail accordingto the second embodiment.

FIGS. 4A and 4B together illustrate a method for measuring thesolvophobic energy and force of the diblock copolymer molecule in thebad solvent to good solvent medium boundary according to the secondembodiment.

FIG. 5A illustrates an example setup for measuring the hydration andsolvophobic force of a biomolecule according to a third embodiment.

FIG. 5B illustrates a graph of the measured force versus molecularextension (F-x) curve according to the third embodiment.

FIG. 6 illustrates a method for measuring the hydration and solvophobicforce of the biomolecule according to the third embodiment.

FIG. 7A illustrates an example setup for measuring the hydration andsolvophobic force of the biomolecule with selected bases attached tolarge hydrophilic molecules according to a fourth embodiment.

FIG. 7B illustrates a graph of the measured force versus molecularextension (F-x) curve according to the fourth embodiment.

FIG. 8 illustrates a method to obtain the position of the modified DNAbases attached to the large hydrophilic molecule according to the fourthembodiment.

FIG. 9 is a method of sequencing a block copolymer according to anembodiment.

FIG. 10 is a method of sequencing a biopolymer/biomolecule according toan embodiment.

FIG. 11 is an example computer (computer setup) having capabilities,which may be included in and/or combined with embodiments.

DETAILED DESCRIPTION

Embodiments of the present invention relate to the field of DNA and RNAsequencing. There is much interest in obtaining the sequence informationof the DNA or RNA molecule with single-molecule based techniques eitherby optical or electrical detection. Examples of these methods includenucleic acid base identification by synthesis and nanopore sequencing.Although various illustrations are provided for DNA and RNA, embodimentscan also be utilized to determine the structural elements of proteinsand sequence them.

The DNA molecule either double-stranded or single-stranded assumes acoil-like shape in solution such as, e.g., water due to its polymericnature. The mechanical properties of a single DNA molecule have beenwidely studied using optical tweezers technique (C. Bustamante, “Tenyears of tension: single-molecule DNA mechanics”, Nature 421, 423-427(2003); K. C. Neuman and S. M. Block, Review of Scientific Instruments75, 2787 (2004)). Each of the two ends of a DNA molecule is attached toa special polymeric bead one of which will be trapped in the opticaltweezers potential well and the other end held by the tip of a glassmicropipette by suction (C. Bustamante et al op.cit.). When the DNAmolecule is stretched it will look like a dumb-bell shape. As the glassmicropipette is pulled away from the optical tweezers slowly with afeed-back controlled nanopositioner, the force being applied on the DNAcan be measured from the displacement of the bead in the optical trapfrom its center with less than 0.1 picoNewton accuracy. These studiesshow that it requires only about 0.1 pico N (10-12 Newtons) to unwindthe DNA from its coil conformation up to a length of ˜0.65 of itscontour length (Lc) and only <5 pN to stretch it to ˜0.85Lc. On applyinga force of ˜65 pN the ds-DNA can be stretched to its full contourlength. Continuous application of this amount of force makes the DNAundergo the overstretching transition.

Atomic force microscope (AFM) is a widely used technique to measure verysmall amount of forces (10-10 to 10-9 Newtons) acting between a surfaceand a sharp tip at the end of a cantilever (further discussed in thefollowing which is herein incorporated by reference: K. C. Neuman and A.Nagy, Nature methods 5, 491 (2008); F. Ritort, Journal of Physics:Condensed Matter, 18, R531 (2006)). The force variation as the tip isscanned over the surface with a nanopositioner can be used to obtainsurface topography and therefore also small molecules bound to thesurface either physically or chemically. By either exploiting thephysical adsorption of the molecules to the surface or their chemicalattachment to the surface groups, one end of the molecule is held to thesurface and the other by the AFM tip. A number of studies have been maderanging from protein folding/unfolding, biomolecular mechanics, theirinteractions with the surface, bond strengths of certain biomolecularinteractions, polymer mechanics, and so forth (“Force Spectroscopy ofPolymers: Beyond Single Chain Mechanics” by X. Zhang, C. Liu and W. Shiin Physical properties of polymers handbook, Chapter 30, Edited by J. E.Mark, 2007, Springer; C. Ortiz and G. Hadziioannou, Macromolecules, 32,780-787 (1999), “Entropic elasticity of single polymer chains ofpoly(methacrylic acid) measured by atomic-force microscopy”; “Singlemolecule force spectroscopy on polysaccharides by atomic forcemicroscopy”, M. Rief, F. Oesterhelt, B. Heymann and H. E. Gaub, Science,275, 1295-1297(1997)).

A charged polymer (such as DNA, RNA, or a protein molecule) that isstretched can become trapped between the gap created in a medium, forexample, an air gap in water. The driving force for this trapping is thesolvation energy gained by the part of the molecule that is exposed toair.

When a point charge moves from a dielectric medium with highpermittivity (∈1) to a low permittivity medium (∈2<∈1), the point chargegains the solvation energy (Born energy) given by:

$\begin{matrix}{\frac{w_{Born}}{kT} = {\frac{q_{0}}{r}\left( {\frac{1}{ɛ\; 2} - \frac{1}{ɛ\; 1}} \right)}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

where q₀=e₀ ²=2 kT=282 Å at room temperature, and e₀ is the value of theelectronic charge.

When the point charge leaves the water-air boundary, the gain insolvation energy amounts to approximately 200 kT, where k is Boltzmann'sconstant and T is temperature. This is equivalent to applying a force ofabout 500 pN (1 pico Newton=10-12 Newton) on the point charge to crossthe boundary. Therefore, if one monitors the force on a charged polymermolecule stretched across a water-air boundary, there will be variationsin the force needed to move the molecule across this boundary dependingon the solvation energy variation for different parts of the molecule.This variation in the force for a charged polymer or a polymer withdifferent types of molecular building blocks as in a diblock copolymercan be used to obtain the information about the position of thedifferent segments along the molecule as explained in detail in theembodiments. This force corresponds to the solvophobic force.

A block copolymer is a polymer consisting of multiple sequences, orblocks, of the same monomer alternating in series with different monomerblocks. The blocks are covalently bound to each other (such as AAABBBAAAfashion, where A and B are two different types of monomers). Blockcopolymers are classified based on the number of blocks they contain andhow the blocks are arranged. For example, block copolymers with twoblocks are called diblocks; those with three blocks are triblocks; andthose with more than three are called multiblocks. The positionaloccurrence of the other blocks can be either in a regular predictableway or can be random as in AAABBBAAABBBAAABBB . . . or AABBBABBAAAAB . .. , respectively. The diblock copolymers in which the second blockoccurs at random positions is called a random diblock copolymer.

Now turning to the figures, FIG. 1A is an example setup 100 formeasuring the hydration and solvophobic force of a polymer strandaccording to an embodiment. One end of the polymer molecule 10 is boundto the bottom surface of the chamber 101 and the other end is bound tothe tip 110 of the atomic force microscope 102. The polymer strand 10can be bound to the either the sample chamber surface or the AFM tip byphysical adsorption or chemical attachment. The polymer strand end canbe covalently linked to a gold surface that acts as the bottom of thechamber through a thiol-modified end group of the polymer. For example,a monofunctional thiol-modified poly-methacrylic acid (PMMA-SH) can besynthesized and has been used in AFM studies of their entropicelasticity (C. Ortiz and G. Hadziioannou, Macromolecules, 32, 780-787(1999), “Entropic elasticity of single polymer chains ofpoly(methacrylic acid) measured by atomic-force microscopy”). In anotherexample of the force spectroscopy studies of Dextran polymer strands,one end of them was attached to a gold surface by epoxy-alkanethiols(“Single molecule force spectroscopy on polysaccharides by atomic forcemicroscopy”, M. Rief, F. Oesterhelt, B. Heymann and H. E. Gaub, Science,275, 1295-1297 (1997)). The other end was attached with streptavidinthrough reaction with carboxymethyl group per glucose unit on averagewhich then bound to the biotin coated Si3N4 AFM tip allowing singlepolymer strand force spectroscopy studies. Further information regardingthe atomic force microscope is discussed below.

Pulling (at a constant rate) the AFM tip 110 (upward in the Z direction)unwinds and stretches the molecule 10 as the tip 110 applies a force onthe molecule 10, all while the whole molecule 10 is still in the goodsolvent medium 115. This force required to expose the segments ofmolecule 10 to the good solvent medium 115 as the molecule 10 unwindsfrom its random coil conformation 20 will result in a plateau (e.g.,Plateau-I in FIG. 1A). FIG. 1B is a graph of the measured force versusmolecular extension (F-x) curve according to the first embodiment inFIG. 1A. While unwinding the molecule 10 from its random coilconformation 20 (i.e., a ball) in the good solvent medium 115 by pullingthe AFM tip 110 upward in the Z direction, the force (designated asforce unwind 1 or Funw1) applied by the AFM tip 110 reaches the firstplateau designated as Plateau-I for the section 103 of the curve.

Further pulling of the AFM tip 110 moves part of the molecule 10 tocross the good solvent medium 115 to a bad solvent medium boundary(i.e., liquid to liquid boundary 120) resulting in the second plateau(i.e., Plateau-II section 104) in the F-x curve. The force to cross theliquid to liquid boundary 120 and move the molecule through the badsolvent medium 125 is designated as force unwind 2 or Funw2. Thedifference between the average force in the two plateau regions (i.e.,between Plateau-I and Plateau-II), δF=Funw1−Funw2, is the solvophobicforce required to move the polymer molecule 10 from the good solventmedium 115 to the bad solvent medium 125. This solvophobic force isdirectly related to the solvophobic energy of the polymer (strand)molecule 10 in the bad solvent with respect to the good solvent. Thepolymer molecule 10 gains solvophobic energy in the bad solvent medium125 compared to the good solvent medium 115. Solvation (also sometimescalled dissolution), is the process of attraction and association ofmolecules of a solvent with molecules or ions of a solute. As ionsdissolve in a solvent, they spread out and surround the solventmolecules.

Pulling the AFM tip 110 (upward in the Z direction) even further so thatthe extended molecule length is almost equal to its contour length Lc(contour length of a polymer is defined as the distance between twomonomers x for the number of monomers that make up the polymer strandwhen the molecule 10 is in fully stretched conformation), the force willexhibit strong non-linear behavior which is related to the elasticproperty of the molecule 10 influenced by its chemical nature. When theAFM tip 110 extends the molecule 10 to its contour length Lc, there is ajump in the (measured) force needed to pull the molecule 10 in the zdirection.

According to the first embodiment, FIG. 2 illustrates a method 200 formeasuring the solvophobic force and solvophobic energy of the polymermolecule 10 in the bad solvent medium 125 with respect to the goodsolvent medium illustrated in FIG. 1. The polymer molecule 10 isfunctionalized with special chemical groups to attach (130, FIG. 1)selectively to the bottom surface 140 of the sample chamber 101 andattach (135, FIG. 1) to the AFM tip 110. The AFM tip 110 is broughtcloser to the bottom surface 140 (of the chamber 101) to attach 135 tothe free end of the polymer molecule 10 that will selectively bind tothe AFM tip at block 201. The AFM tip 110 is pulled (at a constant rate)upward (away from the chamber 101) to unwind and stretch the molecule 10while simultaneously measuring the force required to pull molecule 10while still in the good solvent medium 115 at block 202. Stretching themolecule further (by pulling the AFM tip 110 upward further in the Zdirection) in the good solvent medium 115 will exhibit a plateau region(Plateau-I) in the force-extension curve in FIG. 1B at block 203.

Now, pulling (at a constant rate) the polymer (strand) molecule 10 evenfurther makes the segment of the molecule 10 cross the good solvent-badsolvent boundary 120 near the top of the sample chamber 101; this willresult in a second plateau in the force-extension curve (Plateau-II) atblock 204. At block 205, taking the difference in the average force inPlateau-I and Plateau-II regions is the solvophobic force directlyrelated to the solvophobic energy of the polymer molecule 10 in the badsolvent medium 125 with respect to the good solvent medium 115.

According to a second embodiment, FIG. 3A illustrates the example setup100 for measuring the solvophobic force and energy of a diblock polymerstrand 310 and its nanoscale composition. The nanoscale composition ofthe diblock polymer strand 310 is the length distributions of the two(repeating) blocks 320 and 325 of the polymer that make up the strand.This is equivalent to determining the “polydispersity” of the diblockcopolymer strand 310 at the nanoscale with a precision determined by theAFM measurement setup that can reach about 0.1 nm. However, note thatthe polydispersity of a copolymer as determined by techniques likedynamic light scattering do not obtain the information about positionaloccurrence of the two blocks in either along a single strand, or in thebulk of the polymer. Polydispersity of a copolymer only provides thedistribution of the hydrodynamic radius of the coil-like conformationstaken up by the polymer strands in a solution and the associatedpolydispersity index. One end of the diblock polymer molecule 310 isbound to the bottom surface 140 of the chamber 101 and the other end tothe tip 110 of the atomic force microscope 102. Pulling (at a constantrate) the AFM tip 110 (upward in the Z direction) unwinds and stretchesthe diblock polymer molecule 310 as the tip 110 applies a force on themolecule 310, all while the whole diblock polymer molecule 320 is stillin the good solvent medium 115 (e.g., water). This force required toexpose the segments (e.g., the block 320 and/or block 325) of thediblock polymer molecule 310 to the good solvent medium 115 results in aplateau (Plateau-I in FIG. 3B).

FIG. 3B illustrates a graph of the measured force versus molecularextension (F-x) curve according to the second embodiment. Whileunwinding the molecule 310 from its random coil conformation 20 (i.e., aball) in the good solvent medium 115 by pulling the AFM tip 110 upwardin the Z direction, the force (designated as force unwind 1 or Funw1)applied by the AFM tip 110 reaches the first plateau designated asPlateau-I for the section 303 of the curve.

Further pulling (at a constant rate) of the AFM tip 110 (upward in the Zdirection away from the chamber 101) move one segment (e.g., block 320)of the two blocks 320 and 325 that repeat to make up the diblock polymerstrand 310 to cross the good solvent medium to bad solvent mediumboundary 120 resulting in the second plateau designated as Plateau-II issection 304 in the F-x curve. Pulling tip 110 (upward in the Zdirection) even further will make a segment of the other block 325 ofthe diblock polymer strand 310 to cross the liquid to liquid boundary120. Because the two blocks 320 and 325 have, in general, differentenergies of interaction with the solvent molecules of the good solventmedium 115 and bad solvent medium 125, this results in (either) anadditional drop 355 and/or increase 350 in the force measured by the AFMtip 110 in the second plateau region (Plateau-II in section 304). Theforce-extension curve for the second plateau region therefore appearslike a saw-tooth shape 305. This second plateau region (Plateau-II) isshown in more detail in FIG. 3C. From the positions (identified as XA1,XB1, XA2, XB2 and so on as the molecule 310 is pulled further) obtainedfrom this section 304 of the curve, the nanoscale composition of thediblock copolymer 310 can be obtained. By analogy, this same method canalso be applied to a tri-block and multi-block copolymer to obtain itsnanoscale composition according to the features discussed herein. FIGS.3A, 3B, and 3C may generally be referred to as FIG. 3.

According to the second embodiment, FIGS. 4A and 4B together illustratea method 400 for measuring the solvophobic energy and force of thediblock copolymer molecule 310 in the bad solvent medium 125 withrespect to the good solvent medium 115 illustrated in FIG. 3. Thepolymer molecule 310 is functionalized with special chemical groups toattach 130 selectively to the bottom surface 140 of the sample chamber101 and to attach 135 to the AFM tip 110. Note that in AFM forcespectroscopy studies of elastin-like polypeptides, self-assembledmonolayers of alkanethiols terminated with oligoethylene glycol wereused to graft one end of the polypeptide strands through amine coupling.Amine coupling of the ethylene glycol was carried out by reacting theCOOH groups for 30 minutes with 1-ethyl-3-(dimethylamino) propylcarbodiimide (EDAC) (0.4 M, Aldrich) and N-hydroxysuccinimide(NHS) (0.1M, Aldrich) in Milli-QTMgrade water. One end of the diblock copolymerstrand could also be held by the bottom surface 140 by adsorption andthe other end to the AFM tip 110 by adsorption as well.

Initially, the AFM tip 110 is brought close (e.g., manually orautomatically) to the bottom surface 140 to attach 135 to the free endof the diblock polymer molecule 310 that will selectively bind to theAFM tip at block 401. For example, a chemical is pre-applied to the AFMtip 110, and the same chemical is configured to attach to a block (e.g.,block 320 and/or block 325) of the diblock polymer molecule 310. Thechemical has functionalization properties to both hold/attach to the tip110 (once applied) and likewise attach to the block 320, 325 of thediblock polymer molecule 310 (at the free end not attached to bottomsurface 140).

The AFM tip 110 is pulled (upward in the Z direction) to unwind andstretch the diblock polymer molecule 310, and the AFM 102 measures theforce required at block 402. The AFM tip 110 stretches the diblockpolymer molecule 310 further in the good solvent medium 115 such thatthe measured force exhibits a plateau region of Plateau-I in theforce-extension curve (of FIG. 3) at block 403. Pulling the diblockpolymer (strand) molecule 310 further make the first segment of the twoblocks (such as, e.g., block 320 which is directly attached 135 to theAFM tip 110) (of the diblock copolymer molecule 310) cross the goodsolvent to bad solvent boundary 120 (i.e., the liquid to liquid boundary120) near the top of the sample chamber 101; this results in a secondplateau (Plateau-II) in the force-extension curve of FIG. 3B at block404. The difference between the average force in the first and secondplateau regions gives the solvophobic force of one segment of thediblock copolymer from which the solvation energy can be deduced. Thisis the difference between Funw1 in Plateau-I and Funw2 in Plateau-II.

Pulling the AFM tip 110 upward (in the Z direction) even further makes asegment of the other block (e.g., block 325) of the diblock polymer(strand) molecule 310 to cross the boundary 120 that results inadditional valleys or peaks from the plateau region (Plateau-II). Thepeaks and valleys of the measured force appear as the saw-tooth likevariation (as shown in FIG. 3C) of the pulling force on the AFM tip 110at block 405. The nanoscale composition of the diblock copolymermolecule 310 can be obtained from this saw-tooth shaped force-extensioncurve at block 406. By making many measurements (via the AFM 102) withdifferent strands of the copolymer, average nanoscale composition can beobtained for the polymer sample (diblock polymer molecule 310).

FIG. 5A illustrates an example setup 100 for measuring the hydration andsolvophobic force of a biomolecule 510 like a DNA molecule, RNAmolecule, and/or protein molecule according to a third embodiment. Forexample purposes, the biomolecule is illustrated as the DNA molecule510.

One end of the DNA molecule 510 is bound/attached 130 to the bottomsurface 140 of the chamber 101 and the other end of the DNA molecule 510is attached 135 to the tip 110 of the atomic force microscope 102. TheDNA (or RNA) molecule 510 can be either single-stranded ordouble-stranded. Pulling (at a constant rate) the AFM tip 110 (upward inthe Z direction) unwinds (from the ball 20) and stretches the DNAmolecule 510 as the tip 110 applies a force on the DNA molecule 510,while the whole DNA molecule 510 remains in the good solvent medium 115.This force required to expose the segments (i.e., individual bases shownas shapes on the DNA molecule 510) of the DNA molecule 510 to the goodsolvent medium 115 as the DNA molecule 510 unwinds from its random coilconformation 20 results in a plateau (Plateau-I on section 503 in FIG.5B).

FIG. 5B illustrates a graph of the measured force versus molecularextension (F-x) curve according to the third embodiment. While unwindingthe DNA molecule 510 from its random coil conformation 20 (i.e., a ball)in the good solvent medium 115 by pulling the AFM tip 110 upward in theZ direction, the force (designated as force unwind 1 or Funw1) appliedby the AFM tip 110 reaches the first plateau designated as Plateau-I forthe section 503 of the curve.

Further pulling (at a constant rate) of the AFM tip 110 moves part ofthe DNA molecule 510 to cross the good solvent to bad solvent boundary120 resulting in the second plateau (Plateau-II at section 504) in theF-x curve (as Funw2). The difference between the average force in thetwo plateau regions, δF=Funw1−Funw2, is the solvophobic force requiredto move the DNA or RNA segment (i.e., particular base) from the goodsolvent medium 115 to the bad solvent medium 125. This difference/changein force is directly related to the solvophobic energy of the DNA (orRNA) molecule 510 in the bad solvent medium 125 with respect to the goodsolvent medium 115. The AFM tip 110 (moving upward in the Z direction)pulls the DNA molecule 510 even further so that the extended molecularlength is almost equal to its contour length Lc, the force (on the AFMtip 110) exhibits strong non-linear behavior which is related to theelastic property of the DNA molecule 510 influenced by its chemicalnature. As noted earlier, Lc is the number of monomers x (i.e., bases)for the average separation between the monomers when the DNA molecule510 in stretched conformation.

FIG. 6 illustrates a method 600 for measuring the hydration andsolvophobic force of the biomolecule 510 (e.g., DNA, RNA, or protein).As noted above, the DNA molecule, RNA molecule, or protein molecule issuitably modified at both ends so that one end of the biomolecule 510attaches 130 to the bottom surface 140 of the chamber 101 and the otherend attaches 135 to the AFM tip 110. The operator brings the AFM tip 110close to the bottom surface 140 so as to attach 135 the tip 110 to thefree end of the biomolecule 510, all while the other end of thebiomolecule 510 remains attached 130 to the bottom surface 140 at block601. The operator pulls (at a constant rate) the AFM tip 110 (upward inthe Z direction) to unwind the biomolecule 510 while measuring the forcerequired at block 602. Stretching the molecule further in the goodsolvent medium results in a plateau in the force vs. molecular extensioncurve (shown as Plateau-I in section 503).

Pulling the biomolecule 510 even further (upward in the Z direction)results in the part (e.g., base) of the biomolecule 510 attached to theAFM tip 110 crossing the bad solvent medium 125 region, which results inthe measured force appearing as the second plateau (Plateau-II insection 504) in the F-x curve at block 604. At block 605, the differencebetween the average force in the two plateau regions gives thesolvophobic force required to move to the bad solvent medium 125 fromthe good solvent medium 115.

According to a fourth embodiment, FIG. 7A illustrates an example setup100 for measuring the hydration and solvophobic force of the biomolecule710, such as, e.g., a DNA molecule or RNA molecule attached with largehydrophilic molecules 740 740 through a short oligomer of DNA or RNA.The oligomer binds to the complementary sequence in the DNA or RNAmolecule. The oligomer can consist of 6 to 20 bases of DNA attached withthe large hydrophilic molecule and it would bind to wherever itscomplementary sequence occurs in the long DNA or RNA molecule to besequenced. An example for the biomolecule DNA is the single-strandedviral DNA M13mp18. It is 7249 bases long and its contour length is about5 μm in its fully stretched form. The sequence AATTCCTT occurs at threeplaces separated by about 480 nm and 2.15 μm corresponding to 686 and3066 bases. The complementary oligomers TTAAGGAA attached with biotinmolecule at one end (can be obtained commercially from Midland CertifiedCo., Texas) would bind to the streptavidin coated polystyrene bead whichwill act as a large hydrophilic molecule. The complementary oligomerswould preferentially bind to the single-stranded DNA M13mp18 whereverthe sequence AATTCCTT occurs along the DNA. The hydrophilic nature ofpolystyrene bead and the streptavidin coating would provide the forcevariation necessary to distinguish the location of the oligomer alongthe ssDNA molecule (which then results in distinguishing the sequencesof bases A, T, G, C or U). Further, regarding modification of the DNA orRNA molecule with the large hydrophilic molecule 740 is discussed below.For example purposes, the biomolecule 710 may be referred to as the DNAmolecule 710.

The large hydrophilic molecule 740 attached to the selected segments ofthe DNA molecule 710 through short oligomers produces peaks in themeasured force in Plateau-II (unlike the DNA molecule 510 (in FIG. 5)without the large hydrophilic molecules 740). A hydrophile is a moleculeor other molecular entity that is attracted to and tends to be easilydissolved by water (as would be understood by one of ordinary skill inthe art). An example of the large hydrophilic molecule 740 that may bechemically attached to segments (of the bases) of the DNA molecule 710is polystyrene bead coated with protein molecules Steptavidin.(Commercially available, for example, from Bangs Laboratories Inc,Fishers, Ind. and Invitrogen (Life Technologies), Grand Island, N.Y.).The beads from these sources are available in varying sizes from 30 nmand above. As the large hydrophilic molecule 740 crosses theliquid-liquid boundary, there will be an increase in the measured force750 by the AFM tip 110. Because the position at which these increasesoccur is also measured with a precision of 0.1 nm, this provides theexact positions of the oligomers along the DNA or RNA molecule(correspondingly the position of complementary sequences of bases of theDNA or RNA molecule). As a DNA (or RNA) sequencing method, informationabout the position or location of these modified groups (i.e., oligomersattached to the large hydrophilic molecule 740) along the DNA molecule710 can also be obtained based on the respective measured forcesidentifying and corresponding to different sequences of oligomers of thesame length attached to the large molecule 740. When this is done for alibrary of oligomers (for a word of length N, there are 4N possibleoligomers corresponding to the four bases that each base in the oligomercan take), one can obtain the information about the occurrence of allthese words along the DNA molecule for which the sequence informationneeds to be determined. Therefore, the sequence information about a DNAor RNA molecule can be obtained in this manner. For example, there areoligomers that have various sequences of the bases G, A, T, and C forDNA (where U replaces T for RNA) in the library so that each of the oneoligomer is able to complementary attach to a segment of bases on theDNA molecule 710 be sequenced.

As understood by one skilled in the art complementarity is a propertyshared between two nucleic acid sequences, such that when they arealigned antiparallel to each other, the nucleotide bases at eachposition will be complementary. Two bases are complementary if they formWatson-Crick base pairs. For DNA, adenine (A) bases complement thymine(T) bases and vice versa; guanine (G) bases complement cytosine (C)bases and vice versa. With RNA, it is the same except that uracil ispresent in place of thymine, and therefore adenine (A) bases complementuracil (U) bases. Since there is only one complementary base for each ofthe bases found in DNA and in RNA, one can reconstruct a complementarystrand for any single strand.

Now, returning to the example in FIG. 7A, one end of the DNA molecule710 is bound/attached 130 to the bottom surface 140 of the chamber 101and the other end is bound/attached 135 to the AFM tip 110 of the atomicforce microscope 102. The modified DNA (or RNA) molecule 710 can beeither single-stranded or double-stranded.

The operator-controlled software pulls the AFM tip 110 upward (in the Zdirection at a constant rate), and this unwinds and stretches the DNAmolecule 710 as the AFM tip 110 applies a measured force on the DNAmolecule 710 (all while the whole DNA molecule 710 is still in the goodsolvent medium 115). This force, required to expose the segments (i.e.,bases with and without the attached large molecule 740) of the DNAmolecule 710 to the good solvent medium 115 as the DNA molecule 710unwinds from its random coil conformation 20, results in a plateau(Plateau-I) in the force vs. molecular extension (F-x) curve shown inFIG. 7B.

FIG. 7B illustrates a graph of the measured force versus molecularextension (F-x) curve according to the fourth embodiment. Whileunwinding the DNA molecule 710 from its random coil conformation 20(i.e., a ball) in the good solvent medium 115 by pulling the AFM tip 110upward in the Z direction, the measured force (designated as forceunwind 1 or Funw1) applied by the AFM tip 110 reaches the first plateaudesignated as Plateau-I for the section 703 of the curve.

Further pulling of the AFM tip 110 moves part of the DNA molecule 710 tocross the good solvent medium to bad solvent medium boundary 120resulting in the second plateau (Plateau-II of section 704) in the F-xcurve. Due to the presence of the large hydrophilic molecules 740attached to select oligomers, there are additional jumps in the measuredforce plateau 704 resulting in saw-tooth like shape of theforce-extension curve in this regime (each time the oligomer crosses thegood solvent medium to bad solvent medium boundary 120). As explainedfor the case of determining the nanoscale composition of a diblockcopolymer in FIG. 3, the position (on the DNA molecule 710) of themodified oligomers (i.e., respectively attached to the large hydrophilicmolecule 740) can be obtained from the peak positions (the various peak750 measured forces) in the saw-tooth shape of the force-extension curvein Plateau II shown in FIG. 7B. The repeated peak measured force (peaks750) in the saw-tooth shape (of Plateau II) provides the position of themodified oligomers (respectively attached to the large hydrophilicmolecule 740 on the DNA molecule 710) along with unmodified ones (i.e.,DNA bases not attached to the large hydrophilic molecules 740 withvalley 750 measured forces) is equivalent to sequencing the DNA or RNAmolecule. The sequence information can be used to obtain different typesof genetic information about an individual.

FIG. 8 illustrates a method 800 to obtain the position of the modifiedDNA segments/sequences of bases (i.e., each sequence or segment is agroup of bases) attached to (complementary) oligomers (which areattached to the large hydrophilic molecule 740) along the DNA molecule710 according to the fourth embodiment. Although the oligomers are notshown in FIG. 7A, the oligomer acts as the glue that allows largehydrophilic molecule 740 attach to the segment of bases. Therefore, eachlarge hydrophilic molecule 740 is attached at each segment of bases (andthere may be multiple segments along the DNA molecule 710). Each one ofthese segments (of bases) can be identified via the measured force ofthe large hydrophilic molecule 740. Since the bases of the oligomer areknown in advance, the segment of the bases on the DNA molecule 710 areidentified as the complement to the know bases of the oligomer.

The DNA molecule 710 (or RNA) with the large hydrophilic molecule 740attached to an oligomer is in the sample chamber 101. Initially, one endis attached 130 to the bottom surface 140 of the sample chamber 101 andthe AFM tip 110 is brought closer to the free end (the other end) toattach 135 to the free end of the DNA molecule 710 at block 801.

Pulling (at a constant rate) the AFM tip 110 (upward in the Z direction)unwinds and stretches the DNA molecule 710 while simultaneouslymeasuring the pulling force at block 802, producing Plateau-I (section703) in the force versus extension curve at block 803. As the modifiedDNA molecule 710 (or RNA) crosses the good solvent medium to bad solventmedium boundary 120, the additional (measured) force acting on the AFMtip 110 results in the second plateau (Plateau II in section 704) in theforce at block 804. When the hydrophilic group attached to one type ofbase crosses this liquid to liquid boundary 120, there is an additionalpeak 750 in the plateau II region. This will results in a saw-tooth likevariation of the pulling force on the AFM tip at block 805. The width ofthe dips/valleys 755 and/or peaks 750 in the saw-tooth structureprovides the positional separation between the modified base along theDNA or RNA molecule at block 806.

FIG. 9 is a method 1000 of sequencing a block copolymer (e.g., diblockpolymer strand 310).

The block copolymer (e.g., diblock polymer strand 310) is traversed (viapulling the AFM tip 110 in upward in the z direction at a constant rate)from a first medium (e.g., good solvent medium 115) to a second medium(e.g., bad solvent medium 125) at block 902. The block copolymercomprises first blocks (e.g., blocks 320) and second blocks (e.g.,blocks 325).

The AFM 102 measures a first measured force (e.g., peak 350) when (eachtime) the first blocks traverse from the first medium to the secondmedium at block 904.

At block 906, the AFM 102 measures a second measured force (e.g., valley355) when (each time) the second blocks traverse from the first mediumto the second medium, where the first measured force (e.g., peak 350) isdifferent from the second measured force (e.g., valley 355).

The first blocks of the block copolymer (diblock polymer strand 310) areidentified based on measuring the first measured force (e.g., peaks 350)(each time) via the AFM 102 at block 908, and the second blocks of theblock copolymer are identified based on measuring on the second measuredforce (e.g., valleys 355) at block 910.

In one case, the first measured force is a peak 350 when the firstblocks respectively traverse from the first medium to the second medium,and the second measured force is a valley 355 when the second blocksrespectively traverse from the first medium to the second medium.

The first medium (good solvent medium 115) is water. The second medium(bad solvent medium 125) is oil.

The block copolymer has a first end and a second end. The first end isattached 130 to a bottom surface 140 of a container 101, and the secondend is attached 135 to a tip 110 of a measuring device (AFM 102) thatmeasures forces.

The first measured force is a measured solvophobic force, and the secondmeasured force is a different measured solvophobic force (of the samediblock polymer strand 310).

A length (e.g., lengths XB1, XB2, and so forth) of the first blocks anda location for each of the first blocks are determined based on each ofthe first blocks 325 respectively crossing the good solvent medium 115to bad solvent medium 125 boundary 120. A length (e.g., lengths XA1,XA2, and so forth) of the second blocks and a location of each of thesecond blocks are determined based on each of the second blocks 320respectively crossing the good solvent medium 115 to bad solvent medium125 boundary 120. The first blocks and the second blocks are two typesof monomer blocks.

The locations of the first blocks correspond to the times for the firstmeasured force (e.g., the time for peak 350 repeats), and the locationsof the second blocks correspond to the times for the second measuredforce (e.g., the time for the valley 355 repeats). The location of thefirst blocks and the second blocks respectively along the blockcopolymer is according to each occurrence of the first measured forceand the second force in FIG. 3B.

The block copolymer may be a diblock copolymer, a triblock copolymer,and/or a multiblock copolymer.

FIG. 10 is a method 1000 of sequencing a biopolymer (e.g., biopolymers510 and 710).

The AFM 102 traverses (e.g., by pulling the AFM tip 110 in the zdirection at a constant rate), the biopolymer (e.g., biopolymers 510 and710) from a first medium (e.g., good solvent medium 115) to a secondmedium (e.g., bad solvent medium 125), where the biopolymer comprisesbases at block 1002.

As the biopolymer traverses from the first medium to the second medium,the AFM 102 measures different measured forces (peaks and valleys)corresponding to each of the bases (and/or a group of bases) at block1004.

Based on the different measurement via the AFM 102, the bases (and/or agroup of the different bases) are distinguished from one anotheraccording to the different measured forces measured for each of thebases at block 1006.

Large molecules (e.g., such as different large molecules 740) areattached to selected ones of the bases (e.g., 1, 2, 3, and/or 4 bases)(and/or a group of the bases). The selected base is distinguished by alarge measured force (e.g., valley 750). One or more different largemolecules are respectively attached to the bases (and/or a group ofbases via an oligomer).

Each of the different large molecules is chemically configured to attachto one type of the bases. The AFM tip 110 traverses the biomolecule fromthe first medium to the second medium with the different large moleculesrespectively attached to the different bases causes an increase (i.e.,causes different valleys 750) in the different measured forces measuredfor each of the bases.

The biomolecule may be a DNA molecule, and/or an RNA molecule. The firstmedium (e.g., good solvent medium 115) is water, and the second medium(e.g., bad solvent medium 125) is oil.

More regarding the AFM 102 is discussed below. Atomic force microscopy(AFM) or scanning force microscopy (SFM) is a very high-resolution typeof scanning probe microscopy, with demonstrated resolution on the orderof fractions of a nanometer. The AFM is one of the foremost tools forimaging, measuring, and manipulating matter at the nanoscale. Theinformation is gathered by “feeling” the surface with a mechanicalprobe. Piezoelectric elements that facilitate tiny but accurate andprecise movements on (electronic) command enable the very precisescanning or movement of the AFM tip 110.

The AFM 102 consists of a cantilever with a sharp tip (probe) 110 at itsend that is used to scan or touch the specimen surface. The cantileveris typically silicon or silicon nitride with a tip radius of curvatureon the order of nanometers. When the tip 110 is brought into proximityof a sample surface (e.g., to touch), forces between the tip and thesample lead to a deflection of the cantilever according to Hooke's law.In embodiments, the AFM tip 110 is moved to touch or nearly touch thefree end of the molecule being tested so that the AFM tip 110 attaches135 (chemically and/or physically to the free end of the molecule asdiscussed herein.

Depending on the situation, forces that are measured in AFM includemechanical contact force, van der Waals forces, capillary forces,chemical bonding, electrostatic forces, magnetic forces, Casimir forces,solvation forces, etc. Along with force, additional quantities maysimultaneously be measured through the use of specialized types ofprobes.

The AFM 102 include electronics (such as detectors, etc.) for measuringforces as discussed herein, and understood by one skilled in the art.

Now turning to FIG. 11, an example illustrates a computer 1100 (e.g.,any type of computer system connected to and/or implemented in the AFM102) that may implement features discussed herein. The computer 1100 maybe a distributed computer system over more than one computer. Variousmethods, procedures, modules, flow diagrams, tools, applications,circuits, elements, and techniques discussed herein may also incorporateand/or utilize the capabilities of the computer 1100. Indeed,capabilities of the computer 1100 may be utilized to implement featuresand/or be utilized in conjunction with exemplary embodiments discussedherein in FIGS. 1-10.

Generally, in terms of hardware architecture, the computer 1100 mayinclude one or more processors 1110, computer readable storage memory1120, and one or more input and/or output (I/O) devices 1170 that arecommunicatively coupled via a local interface (not shown). The localinterface can be, for example but not limited to, one or more buses orother wired or wireless connections, as is known in the art. The localinterface may have additional elements, such as controllers, buffers(caches), drivers, repeaters, and receivers, to enable communications.Further, the local interface may include address, control, and/or dataconnections to enable appropriate communications among theaforementioned components.

The processor 1110 is a hardware device for executing software that canbe stored in the memory 1120. The processor 1110 can be virtually anycustom made or commercially available processor, a central processingunit (CPU), a data signal processor (DSP), or an auxiliary processoramong several processors associated with the computer 1100, and theprocessor 1110 may be a semiconductor based microprocessor (in the formof a microchip) or a macroprocessor.

The computer readable memory 1120 can include any one or combination ofvolatile memory elements (e.g., random access memory (RAM), such asdynamic random access memory (DRAM), static random access memory (SRAM),etc.) and nonvolatile memory elements (e.g., ROM, erasable programmableread only memory (EPROM), electronically erasable programmable read onlymemory (EEPROM), programmable read only memory (PROM), tape, compactdisc read only memory (CD-ROM), disk, diskette, cartridge, cassette orthe like, etc.). Moreover, the memory 1120 may incorporate electronic,magnetic, optical, and/or other types of storage media. Note that thememory 1120 can have a distributed architecture, where variouscomponents are situated remote from one another, but can be accessed bythe processor(s) 1110.

The software in the computer readable memory 1120 may include one ormore separate programs, each of which comprises an ordered listing ofexecutable instructions for implementing logical functions. The softwarein the memory 1120 includes a suitable operating system (0/S) 1150,compiler 1140, source code 1130, and one or more applications 1160 ofthe exemplary embodiments. As illustrated, the application 1160comprises numerous functional components for implementing the features,processes, methods, functions, and operations of the exemplaryembodiments.

The operating system 1150 may control the execution of other computerprograms, and provides scheduling, input-output control, file and datamanagement, memory management, and communication control and relatedservices.

The application 1160 may be a source program, executable program (objectcode), script, or any other entity comprising a set of instructions tobe performed. When a source program, then the program is usuallytranslated via a compiler (such as the compiler 1140), assembler,interpreter, or the like, which may or may not be included within thememory 1120, so as to operate properly in connection with the O/S 1150.Furthermore, the application 1160 can be written as (a) an objectoriented programming language, which has classes of data and methods, or(b) a procedure programming language, which has routines, subroutines,and/or functions.

The I/O devices 1170 may include input devices (or peripherals) such as,for example but not limited to, a mouse, keyboard, scanner, microphone,camera, etc. Furthermore, the I/O devices 1170 may also include outputdevices (or peripherals), for example but not limited to, a printer,display, etc. Finally, the I/O devices 1170 may further include devicesthat communicate both inputs and outputs, for instance but not limitedto, a NIC or modulator/demodulator (for accessing remote devices, otherfiles, devices, systems, or a network), a radio frequency (RF) or othertransceiver, a telephonic interface, a bridge, a router, etc. The I/Odevices 1170 also include components for communicating over variousnetworks, such as the Internet or an intranet. The I/O devices 1170 maybe connected to and/or communicate with the processor 1110 utilizingBluetooth connections and cables (via, e.g., Universal Serial Bus (USB)ports, serial ports, parallel ports, FireWire, HDMI (High-DefinitionMultimedia Interface), etc.).

In exemplary embodiments, where the application 1160 is implemented inhardware, the application 1160 can be implemented with any one or acombination of the following technologies, which are each well known inthe art: a discrete logic circuit(s) having logic gates for implementinglogic functions upon data signals, an application specific integratedcircuit (ASIC) having appropriate combinational logic gates, aprogrammable gate array(s) (PGA), a field programmable gate array(FPGA), etc.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of onemore other features, integers, steps, operations, element components,and/or groups thereof.

The corresponding structures, materials, acts, and equivalents of allmeans or step plus function elements in the claims below are intended toinclude any structure, material, or act for performing the function incombination with other claimed elements as specifically claimed. Thedescription of the present invention has been presented for purposes ofillustration and description, but is not intended to be exhaustive orlimited to the invention in the form disclosed. Many modifications andvariations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the invention. Theembodiment was chosen and described in order to best explain theprinciples of the invention and the practical application, and to enableothers of ordinary skill in the art to understand the invention forvarious embodiments with various modifications as are suited to theparticular use contemplated

The flow diagrams depicted herein are just one example. There may bemany variations to this diagram or the steps (or operations) describedtherein without departing from the spirit of the invention. Forinstance, the steps may be performed in a differing order or steps maybe added, deleted or modified. All of these variations are considered apart of the claimed invention.

While the preferred embodiment to the invention had been described, itwill be understood that those skilled in the art, both now and in thefuture, may make various improvements and enhancements which fall withinthe scope of the claims which follow. These claims should be construedto maintain the proper protection for the invention first described.

What is claimed is:
 1. A method of sequencing a biopolymer, comprising:traversing the biopolymer from a first medium to a second medium;wherein the biopolymer comprises bases; as the biopolymer traverses fromthe first medium to the second medium, measuring different measuredforces corresponding to each of the bases; and distinguishing the basesfrom one another according to the different measured forces measured foreach of the bases.
 2. The method of claim 1, further comprisingattaching large molecules to selected ones of the bases.
 3. The methodof claim 2, further comprising distinguishing the selected ones of thebases by a large measured force.
 4. The method of claim 1, furthercomprising attaching one or more different large molecules respectivelyto the bases.
 5. The method of claim 4, wherein each of the differentlarge molecules are chemically configured to attach to one type of thebases.
 6. The method of claim 5, further comprising traversing thebiopolymer from the first medium to the second medium with the differentlarge molecules respectively attached to the bases causes an increase inthe different measured forces measured for each of the bases.
 7. Themethod of claim 1, wherein the biopolymer is a DNA molecule.
 8. Themethod of claim 1, wherein the biopolymer is an RNA molecule.
 9. Themethod of claim 1, wherein the first medium is water; and wherein thesecond medium is oil.
 10. An apparatus for sequencing a biopolymer, theapparatus comprising: a chamber filled with a first medium and a secondmedium; and an atomic force microscope; wherein the atomic forcemicroscope is configured to: traverse the biopolymer from the firstmedium to the second medium, wherein the biopolymer comprises bases; asthe biopolymer traverses from the first medium to the second medium,measure different measured forces corresponding to each of the bases;and distinguish the bases from one another according to the differentmeasured forces measured for each of the bases.
 11. The apparatus ofclaim 10, wherein large molecules are attached to selected ones of thebases.
 12. The apparatus of claim 11, wherein the selected ones of thebases are distinguished by a large measured force.
 13. The apparatus ofclaim 10, wherein one or more different large molecules are attachedrespectively to the bases.
 14. The apparatus of claim 13, wherein eachof the different large molecules are chemically configured to attach toone type of the bases.
 15. The apparatus of claim 14, further comprisingtraversing the biopolymer from the first medium to the second mediumwith the different large molecules respectively attached to the basescauses an increase in the different measured forces measured for each ofthe bases.
 16. The apparatus of claim 10, wherein the biopolymer is aDNA molecule.
 17. The apparatus of claim 10, wherein the biopolymer isan RNA molecule.
 18. The apparatus of claim 10, wherein the first mediumis water; and wherein the second medium is oil.